What is the difference between a buffalo and a bison?*

The question of how a genotype (the arrangement of letters in DNA) maps to a phenotype (the shape and behavior of an organism) can be examined at many levels. On the one hand, we’d like to know how small differences in DNA sequence determine differences between individual humans, such as susceptibility to disease, height, IQ, maybe musical talent… the list is long. On the other hand, we’d like to know which DNA differences determine the crucial differences between species. What makes the beak short or long or pointy, what makes the neck short or long, what determines the size of the average member of the species? Many of these factors can vary significantly within a species, too: take, for example, the enormous range of size that we see in dogs, from Elwood the 9cm-high Jack Russell to Giant George the 110cm-tall Great Dane. While some of these differences are at least partly understood — size variation in dogs has been traced to a polymorphism in the gene for the growth hormone IGF-1, for example — there are many many others that we simply don’t understand.

One of the surprising findings of the molecular age, which has become even clearer as genome sequence becomes more available, is that changes in protein coding sequences are less prevalent than changes in sequences that may affect the regulation of a gene. This is rather irritating because (as we’ve discussed before) it’s not easy to use sequence information to predict how the expression of a gene might change. So there’s not a great deal of information on whether and when different species use the same gene (or set of genes) at different times, or whether and when a gene is expressed to a higher level in one species relative to another. But some of the differences between species are almost certainly encoded in the genome in this way. There’s been particular interest in changes in timing, called heterochrony, going back to seminal discoveries in the 1980’s about heterochronic genes in C. elegans that affect the timing of developmental events. But we don’t know how frequent such changes are.

A recent paper (Yanai et al. 2011. Mapping gene expression in two Xenopus species: Evolutionary constraints and developmental flexibility. Developmental CellPMID: 21497761) sets out to survey differences in gene expression between two different species of frog, Xenopus laevis and Xenopus tropicalis. These frogs are similar in some ways — they have fat little back legs and an egg-shaped body, for example — but very different in others. X. laevis is about twice the size of X. tropicalis, and takes about 4x as long to grow to maturity. X. tropicalis prefers higher temperatures than X. laevis for development (28° C vs. 22°C). On the molecular level, the largest difference is that X. laevis is tetraploid, with more or less 4 copies of every gene (presumably because two diploid cells accidentally fused), while X. tropicalis is diploid. This makes X. tropicalis a better model for genetic studies, while X. laevis is the favored organism for biochemical studies due to the large size of its eggs (1 mm). The most recent common ancestor for the two species lived at least 30 million years ago, so the genome sequences have undoubtedly diverged significantly: the X. tropicalis genome came out last year, but the X. laevis genome is not yet complete, so we don’t know the details of the sequence differences.

The nice thing about these two frogs is that they go through almost identical developmental stages. If you leave aside the little matter of size, both frog embryos look identical throughout the blastula, gastrula, neurula and tailbud stages and sub-stages. In fact, the same table of images is used for both frogs to determine what stage in development they’ve reached. This is important if you want to track changes in gene expression: detecting changes in timing of expression requires some kind of baseline to compare the pattern of gene expression to, and even changes in level of expression can be misinterpreted if the relative timing is unclear. Yanai et al. took carefully staged embryos and measured the RNA levels transcribed from over ten thousand genes at different developmental time points. They did this by mashing up one X. laevis embryo or three X. tropicalis embryos (to get similar amounts of RNA from both species), spiking in foreign RNAs at known concentrations, and measuring RNA levels using custom-designed microarrays carefully designed to make expression levels in one species comparable to the expression levels in the other.

There is a useful tool on the Kirschner lab webpage that allows you to view all of the results from the study: you can put in your own favorite gene name and see how the level of transcription of that gene changes by developmental stage in the two frogs. Overall, the pattern of transcription is very similar in the two species, but there are also some differences. Many of these will take a significant amount of time and work to sort out, but here are some of the changes that the authors picked out as potentially easy to rationalize:

• The components of the membrane attack complex of the complement cascade are induced much earlier in X. tropicalis than in X. laevis. I must say that this was a big surprise to me when I first heard about it: this is supposed to be a study of development, not of the immune system, after all. But actually it makes sense, if you assume that the warmer waters X. tropicalis grows in expose the developing embryos to more bacteria. The complement system (a beautiful bit of biochemistry that is not nearly as famous as it should be) is a major bulwark in the body’s defenses against bacteria, especially Gram-negative bacteria, and it’s plausible that earlier expression of complement genes would be selected in warmer but more infectious waters.

• Similarly, hemoglobin genes and another gene that also encodes a protein involved in oxygen transport are expressed earlier in X. tropicalis. Since warmer waters carry less oxygen, this makes sense too.

• X. tropicalis hatches at an earlier developmental stage than X. laevis, and as you would expect, the hatching enzymes that dissolve the envelope around the embryo are transcribed earlier for tropicalis than for laevis.

• The timing of the expression of transcription factors is very well conserved between the two species, but the timing of the expression of signaling pathway components shows more variation. This is odd, because usually a signaling pathway is what triggers the function of a transcription factor. Possibly this indicates that it’s relatively easy to make changes in the connections between signaling pathways and transcription factors. The same transcription factors may be present at the same developmental stages, with different signaling pathways being used to activate them.

Although these changes in timing are interesting, there are not all that many of them. Changes in the level to which different genes are expressed are more frequent, and conservation is the dominant theme. Could this mean that the differences that lead to important changes will be relatively easy to pin down? (Well, we can hope…. )

Leon Peshkin, one of the authors, likes to think of the difference between the two organisms as something like what happens when two musicians playing different instruments interpret the same musical score. A section marked allegro might be taken a little faster by the violin than the cello, perhaps; and if you tell both instruments to play an A, the result will be different in pitch, although the sequence AGCA will be recognizably the same sequence no matter which instrument plays it. (T is more difficult to fit into a musical analogy). Leon went so far as to sketch his vision of a frog duet for the cover of the journal that published the paper — sadly, the journal decided to use something a little more ordinary, so I reproduce Leon’s sketch here for your amusement and edification.

* You can’t wash your hands in a buffalo. **

** Only funny when delivered in a strong Brummie accent, in which bison = basin.